Over 90% of the population harbor EBV-latently infected cells. After a typically benign primary infection of B cells, EBV transitions to a latent state in which the virus expresses few viral transcripts to avoid immune recognition. These latently-infected cells are believed to be the precursors for EBV-associated lymphomas, including Burkitt's lymphoma, Hodgkin's and Non-Hodgkin's lymphoma. An EBV protein that is expressed during latency and by EBV-associated tumors is Latent Membrane Protein 2A (LMP2A). LMP2A enhances B cell survival and accelerates tumor onset. Preliminary data for this proposal indicate that LMP2A enhances mitogen-induced B cell proliferation and the production of IL-10, which is a cytokine that promotes B cell proliferation and survival. Therefore, LMP2A-mediated increases in IL-10 production may impact the maintenance of viral latency and the generation of EBV-associated lymphomas. The hypothesis for this proposal is that LMP2A enhances B cell proliferation by increasing B cell IL-10 production. Experiments using both LMP2A-expressing and LMP2A-negative cell lines, in addition to B cells derived from murine LMP2A transgenic mouse models will test this hypothesis. In Aim 1 we will use quantitative Realtime RT-PCR, ELISpot, and intracellular IL-10 staining to confirm that LMP2A increases mitogen-induced IL-10 production. In Aim 2 we will use cell lines containing wildtype LMP2A and mutant forms of LMP2A to determine the residues of the cytoplasmic tail of LMP2A required to increase IL-10 production. Additionally, we will use pharmacologic inhibitors that inactivate targets of LMP2A to determine the proteins required to increase IL-10 production. Finally, in Aim 3 we will inactivate the IL-10 signaling pathway using a neutralizing anti-IL-10 antibody and inhibitors specific for the IL-10 receptor signal transduction pathway to determine if LMP2A-dependent increases in IL-10 mediate the hyperproliferation of LMP2A-expressing B cells. Understanding the mechanisms by which LMP2A increases B cell proliferation and IL-10 production will advance our knowledge of how LMP2A maintains EBV latency and promotes EBV-associated cancers. PUBLIC HEALTH RELEVANCE: Over 90% of the population harbor B cells latently infected with Epstein-Barr Virus (EBV) that is associated with the development of lymphomas, such as Burkitt's lymphoma, Hodgkin's lymphoma and Non-Hodgkin's lymphoma. This proposal will expand our understanding of how EBV increases the production of the cytokine IL-10 to increase proliferation and survival of B cells. Findings from these studies will not only help us to understand the biology of EBV, but also to identify mechanisms by which EBV promotes tumor development.